Anthracnose Resistant Alfalfa Plants

ABSTRACT

The present disclosure provides alfalfa plants exhibiting broad spectrum resistance to Race 1, Race 2, and Race 5 anthracnose. Such plants may comprise novel introgressed genomic regions associated with disease resistance from Race 1, Race 2, and Race 5 anthracnose. In certain aspects, compositions, including novel polymorphic markers and methods for producing, breeding, identifying, and selecting plants or germplasm with a disease resistance phenotype are provided. Also provided are alfalfa varieties designated as C0416C4164 and H0415C4114. Provided by the invention are the seeds, plants and derivatives of alfalfa varieties C0416C4164 and H0415C4114. Also provided by the invention are tissue cultures of alfalfa varieties C0416C4164 and H0415C4114, and the plants regenerated therefrom. Still further provided by the invention are methods for producing alfalfa plants by crossing alfalfa variety C0416C4164 or H0415C4114 with itself or another alfalfa variety and plants produced by such methods.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/532,151, filed Jul. 13, 2017, which is herein incorporated byreference in its entirety.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “LLKS004US_ST25.txt” whichis 9.80 kilobytes (measured in MS-Windows®) and created on Jul. 13,2018, and comprises 7 sequences, is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture and morespecifically to methods and compositions for producing alfalfa plantsexhibiting improved resistance to anthracnose (Colletotrichum trifolii).

BACKGROUND OF THE INVENTION

Disease resistance is an important trait in agriculture, particularly ina crop such as alfalfa. Alfalfa can be grown in wide range of climatesand ecosystems and is one of the United States most valuablefood-production related crops. Anthracnose in alfalfa plants, caused byColletotrichum trifolii, is one of the most serious diseases affectingalfalfa and results in stem death, crown rot, and reduced wintersurvival. Alfalfa resistant to one race of anthracnose may not beresistant to another race of anthracnose. New alfalfa lines comprisingresistance to one or more races of anthracnose are needed given thesignificant importance of this disease in crop production.

SUMMARY OF THE INVENTION

One aspect of the invention provides a Medicago sativa plant comprisingan introgressed allele conferring to said plant increased broad-spectrumresistance to Colletotrichum trifolii Race 5 compared with a plant notcomprising said allele, wherein a representative sample of seedcomprising said allele has been deposited under ATCC Accession No.PTA-124210 or under ATCC Accession No. PTA-125043. In some aspects, theallele is partially dominant. In other aspects, the introgressed allelecomprises the resistance haplotype found in H0415C4112, H0415C4114,H0415C4115, or H0415C4116. The invention also relates to anthracnoseresistant Medicago sativa plants, wherein the broad-spectrum resistanceto Colletotrichum trifolii confers resistance to Colletotrichum trifoliiRaces 1, 2, and 5. The invention also relates to anthracnose resistantMedicago sativa plants, wherein said broad-spectrum resistance toColletotrichum trifolii confers resistance to Colletotrichum trifoliiRace 5. Further provided are seeds that produce a Medicago sativa plantcomprising an introgressed allele conferring to said plant increasedbroad-spectrum resistance to Colletotrichum trifolii Race 5 comparedwith a plant not comprising said allele wherein a representative sampleof seed comprising said allele has been deposited under ATCC AccessionNo. PTA-124210 or under ATCC Accession No. PTA-125043. Further providedare plant parts, including cells, seeds, roots, stems, leaves, heads,flowers, or pollen. Also provided are progeny plants of the Medicagosativa plants described herein.

Another aspect of the invention provides method for producing a Medicagosativa plant with broad-spectrum resistance to Colletotrichum trifolii,comprising the steps of: a) crossing the plant of claim 1 with itself orwith a Medicago sativa plant of a different genotype to produce one ormore progeny plants; and b) selecting a progeny plant comprising saidallele. In further aspects, the selecting said progeny plant comprisesidentifying a genetic marker genetically linked to said allele. In otheraspects of the method, the selecting a progeny plant comprisesidentifying a genetic marker within or genetically linked to a genomicregion flanked in the genome of said plant by first identified markerlocus and second identified marker locus. In yet further aspects of themethod, the selecting a progeny plant comprises detecting at least onepolymorphism at a locus selected from the group consisting of firstmarker locus, second marker locus, and third marker locus. In otheraspects of the method, the progeny plant is an F2-F6 progeny plant. Infurther aspects of the method, the producing said progeny plantcomprises backcrossing. In other aspects of the method, backcrossingcomprises from 2-7 generations of backcrossing. In yet another aspect,the method provides a plant produced by the method.

The invention also relates to a plant of alfalfa variety C0416C4164,representative seed of said alfalfa variety having been deposited underATCC Accession No. PTA-124210. Also provided is a plant part of theplant of alfalfa variety C0416C4164, wherein the plant part comprises atleast one cell of said plant.

In a further aspect, provided is a seed of alfalfa variety C0416C4164,wherein representative seed of said alfalfa variety have been depositedunder ATCC Accession No. PTA-124210. Also provided is a method ofproducing alfalfa seed, the method comprising crossing the plant of theseed of alfalfa variety C0416C4164, wherein representative seed of saidalfalfa variety have been deposited under ATCC Accession No. PTA-124210,with itself or a second alfalfa plant to produce said alfalfa seed. Infurther aspects, the method further comprising crossing the plant ofalfalfa variety C0416C4164 with a second, nonisogenic alfalfa plant toproduce said alfalfa seed. In yet further aspect, an F₁ alfalfa seedproduced by the method of producing alfalfa seed. Also provided is analfalfa plant produced by growing the F₁ alfalfa seed.

Another aspect of the invention relates to a composition comprising theseed of alfalfa variety C0416C4164, wherein representative seed of saidalfalfa variety have been deposited under ATCC Accession No. PTA-124210,further comprised in plant seed growth media. In further aspects, theplant seed growth media is soil or a synthetic cultivation medium.

In a further aspect, provided is a method of producing alfalfa seed, themethod comprising crossing the plant of the seed of alfalfa varietyC0416C4164, wherein representative seed of said alfalfa variety havebeen deposited under ATCC Accession No. PTA-124210, with itself or asecond alfalfa plant to produce said alfalfa seed, wherein the methodfurther comprising: (a) crossing a plant grown from said alfalfa seedwith itself or a different alfalfa plant to produce seed of a progenyplant of a subsequent generation; (b) growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant to produce seed of a progeny plant of afurther subsequent generation; and (c) repeating step (b) withsufficient inbreeding to produce seed of an inbred alfalfa plant that isderived from alfalfa variety C0416C4164. In other aspects, the methodfurther comprises crossing a plant grown from said seed of an inbredalfalfa plant that is derived from alfalfa variety C0416C4164 with anonisogenic plant to produce seed of a hybrid alfalfa plant that isderived from alfalfa variety C0416C4164.

The invention also relates to a method of producing a commodity plantproduct, the method comprising producing the commodity plant productfrom a plant of alfalfa variety C0416C4164, representative seed of saidalfalfa variety having been deposited under ATCC Accession No.PTA-124210. In further aspects, the method produces a commodity plantproduct, wherein the commodity plant product comprises at least one cellof alfalfa variety C0416C4164.

The invention also relates to a plant of alfalfa variety H0415C4114,representative seed of said alfalfa variety having been deposited underATCC Accession No. PTA-125043. Also provided is a plant part of theplant of alfalfa variety H0415C4114, wherein the plant part comprises atleast one cell of said plant.

In a further aspect, provided is a seed of alfalfa variety H0415C4114,wherein representative seed of said alfalfa variety have been depositedunder ATCC Accession No. PTA-125043. Also provided is a method ofproducing alfalfa seed, the method comprising crossing the plant of theseed of alfalfa variety H0415C4114, wherein representative seed of saidalfalfa variety have been deposited under ATCC Accession No. PTA-125043,with itself or a second alfalfa plant to produce said alfalfa seed. Infurther aspects, the method further comprising crossing the plant ofalfalfa variety H0415C4114 with a second, nonisogenic alfalfa plant toproduce said alfalfa seed. In yet further aspect, an F₁ alfalfa seedproduced by the method of producing alfalfa seed. Also provided is analfalfa plant produced by growing the F₁ alfalfa seed.

Another aspect of the invention relates to a composition comprising theseed of alfalfa variety H0415C4114, wherein representative seed of saidalfalfa variety have been deposited under ATCC Accession No. PTA-125043,further comprised in plant seed growth media. In further aspects, theplant seed growth media is soil or a synthetic cultivation medium.

In a further aspect, provided is a method of producing alfalfa seed, themethod comprising crossing the plant of the seed of alfalfa varietyH0415C4114, wherein representative seed of said alfalfa variety havebeen deposited under ATCC Accession No. PTA-125043, with itself or asecond alfalfa plant to produce said alfalfa seed, wherein the methodfurther comprising: (a) crossing a plant grown from said alfalfa seedwith itself or a different alfalfa plant to produce seed of a progenyplant of a subsequent generation; (b) growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant to produce seed of a progeny plant of afurther subsequent generation; and (c) repeating step (b) withsufficient inbreeding to produce seed of an inbred alfalfa plant that isderived from alfalfa variety H0415C4114. In other aspects, the methodfurther comprises crossing a plant grown from said seed of an inbredalfalfa plant that is derived from alfalfa variety H0415C4114 with anonisogenic plant to produce seed of a hybrid alfalfa plant that isderived from alfalfa variety H0415C4114.

The invention also relates to a method of producing a commodity plantproduct, the method comprising producing the commodity plant productfrom a plant of alfalfa variety H0415C4114, representative seed of saidalfalfa variety having been deposited under ATCC Accession No.PTA-125043. In further aspects, the method produces a commodity plantproduct, wherein the commodity plant product comprises at least one cellof alfalfa variety H0415C4114.

Another aspect of the invention also relates to a Medicago sativa plantcomprising a recombinant chromosomal segment on chromosome 4, whereinsaid chromosomal segment comprises an introgressed Colletotrichumtrifolii race 5 resistance allele conferring to said plant increasedresistance to Colletotrichum trifolii race 5 compared to a plant notcomprising said allele. In some aspects, the Colletotrichum trifoliirace 5 resistance allele is located within a chromosomal segment isflanked by marker locus FG2208 (SEQ ID NO: 1) and marker locus FG27271(SEQ ID NO: 7) on chromosome 4 in said plant. In other aspects, therecombinant chromosomal segment comprises a marker locus selected fromthe group consisting of FG2208 (SEQ ID NO: 1), FG2218 (SEQ ID NO: 2),FG27062 (SEQ ID NO: 3), FG2226 (SEQ ID NO: 4), FG27232 (SEQ ID NO: 5),FG27251 (SEQ ID NO: 6), and FG27271 (SEQ ID NO: 7) on chromosome 4. Inother aspects the Colletotrichum trifolii race 5 resistance allele islocated within a chromosomal segment in the genome of said plant flankedby 16,945,124 bp to 19,292,176 bp on the Medicago truncatula Mt4.0 map.In yet a further aspect, a sample of seed comprising said introgressedColletotrichum trifolii race 5 resistance allele was deposited underATCC Accession No. PTA-124210 or under ATCC Accession No. PTA-125043.

Another aspect of the invention relates to a method for producing aMedicago sativa plant with broad-spectrum resistance to Colletotrichumtrifolii, comprising the steps of: a) crossing the plant of claim 44with itself or with a Medicago sativa plant of a different genotype toproduce one or more progeny plants; and b) selecting a progeny plantcomprising said allele. In one aspect, the selecting a progeny plantcomprises detecting said allele flanked by marker locus FG2208 (SEQ IDNO: 1) and marker locus FG27271 (SEQ ID NO: 7). In another aspect, theprogeny plant is an F₂-F₆ progeny plant. In a further aspect, theproducing said progeny plant comprises backcrossing.

The invention also relates to a method of producing a Medicago sativaplant exhibiting resistance to Colletotrichum trifolii race 5,comprising introgressing into a plant a Colletotrichum trifolii race 5resistance allele within a recombinant chromosomal segment flanked inthe genome of said plant by marker locus FG2208 (SEQ ID NO: 1) andmarker locus FG27271 (SEQ ID NO: 7) on chromosome 4, wherein said alleleconfers increased resistance to Colletotrichum trifolii race 5 comparedto a plant not comprising said allele. In one aspect, the recombinantchromosomal segment comprises a marker locus selected from the groupconsisting of FG2208 (SEQ ID NO: 1), FG2218 (SEQ ID NO: 2), FG27062 (SEQID NO: 3), FG2226 (SEQ ID NO: 4), FG27232 (SEQ ID NO: 5), FG27251 (SEQID NO: 6), and FG27271 (SEQ ID NO: 7). In another aspect, saidintrogressing comprises backcrossing. In a further aspect, saidintrogressing comprises marker-assisted selection. In another aspect,said introgressing comprises assaying said plant for increasedColletotrichum trifolii race 5 resistance. In yet another aspect, theinvention provides a Medicago sativa obtainable by the methods disclosesherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Illustrates anthracnose race 5 resistance ratings forexperimental varieties that were developed for race 5 anthracnoseresistance.

FIG. 2: Illustrates an experiment comparing source founder populationfor race 5 anthracnose (RRL43A132) to derived populations (Synthetics)which are a subset of the experimental varieties that were developed forrace 5 anthracnose resistance. A rating of 0 indicates plant dead and arating of 4 indicates significant resistant. The graph shows thedistribution of individual plants within the population. Thesepopulations were subject to 1 cycle of recurrent selection for race 5anthracnose resistance. Plants scored as a 3 or 4 are consideredresistant.

FIG. 3A: Illustrates that all RRL43A132 alfalfa 13 days afterinoculation with Race 5 anthracnose, are dead. RRL43A132 is the sourcepopulation that led to many of the derived anthracnose Race 5 resistantexperimental plants.

FIG. 3B: Illustrates that H0416C4116 alfalfa 13 days after inoculationwith Race 5 anthracnose yielded a large percentage of survivors comparedto the founder population (this population has had 1 cycle of phenotypicselection for Race 5 anthracnose resistance).

DETAILED DESCRIPTION

Anthracnose (Colletrotrium trifolii) was first identified as animportant alfalfa pathogen in the mid-late 1960's in the mid-Atlanticregion of the U.S. Over the next decade anthracnose became one of themost damaging diseases in alfalfa over the entire eastern half of thecountry. An intensive breeding program at USDA Beltsville resulted inthe commercial release of the anthracnose resistant variety Arc in 1974.By 1979 a new race of anthracnose had developed that overwhelmed theresistance gene An1 found in Arc. Although additional sources ofresistance to anthracnose were subsequently developed, the rapidemergence of new races has made this disease a continued cause ofsignificant crop loss. The present invention represents a significantadvance in that it provides alfalfa plants resistant to newly identifiedRace 5 anthracnose, as well as Race 1 and Race 2.

I. GENOMIC REGIONS, ALLELES, AND POLYMORPHISMS ASSOCIATED WITHANTHRACNOSE RESISTANCE IN ALFALFA PLANTS

Anthracnose is a harmful alfalfa pathogen that typically kills orseriously injures alfalfa plants, such that infection with anthracnosecan significantly and deleteriously impact crop yield of the plant. Itis therefore desirable to identify specific genomic regions conferringresistance to specific races of anthracnose or broad-spectrum resistanceto several races. Although intensive efforts to develop such resistancehave been undertaken, previous efforts to develop new anthracnoseresistant lines have met with limited success due to complicatingfactors such as linkage drag resulting in resistant lines withunacceptable agronomic quality. Further, in view of the emergence of newanthracnose races, new and durable resistance alleles are needed.Despite these obstacles, the present inventors have identified newsources of broad-spectrum anthracnose resistance and developed novelplant varieties comprising these resistance alleles.

The invention provides novel plant varieties along with novelintrogressions of one or more alleles associated with increasedanthracnose race 5 resistance in alfalfa plants, together withpolymorphic nucleic acids and linked markers for tracking theintrogressions during plant breeding. The novel plant varieties may beused together with the novel trait-linked markers provided herein inaccordance with certain embodiments of the invention. In certainembodiments, the invention provides alfalfa plants comprising donor DNAfrom an anthracnose race 5 resistant line between marker locus FG2208(SEQ ID NO: 1) and FG27271 (SEQ ID NO: 7) on chromosome 4.

One of skill in the art will understand that interval values may varybased on factors such as the reference map that is used, the sequencingcoverage and the assembly software settings. However, such parametersand mapping protocols are known in the art and one of skill in the artcan use the marker sequences provided herein to physically andgenetically anchor the introgressions described herein to any given mapusing such methodology. The novel introgression of the present inventionconfers unique significantly improved agronomic properties overpreviously disclosed anthracnose resistance introgressions.

II. INTROGRESSION OF GENOMIC REGIONS ASSOCIATED WITH DISEASE RESISTANCE

Marker-assisted introgression involves the transfer of a chromosomalregion defined by one or more markers from a first genetic background toa second. Offspring of a cross that contain the introgressed genomicregion can be identified by the combination of markers characteristic ofthe desired introgressed genomic region from a first genetic backgroundand both linked and unlinked markers characteristic of the secondgenetic background.

The present invention provides novel markers for identifying andtracking introgression of one or more of the genomic regions from analfalfa into cultivated alfalfa lines. The invention further providesmarkers for identifying and tracking the novel introgressions disclosedherein during plant breeding, including markers relevant for identifyingalfalfa plants with the desired Race 5 anthracnose resistance trait.

Markers within or linked to any of the genomic intervals of the presentinvention can be used in a variety of breeding efforts that includeintrogression of genomic regions associated with disease resistance intoa desired genetic background. For example, a marker within 20 cM, 15 cM,10 cM, ScM, 2 cM, or 1 cM of a marker associated with disease resistancedescribed herein can be used for marker-assisted introgression ofgenomic regions associated with a disease tolerant phenotype.

Alfalfa plants comprising one or more introgressed regions associatedwith a desired phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or99% of the remaining genomic sequences carry markers characteristic ofthe germplasm are also provided. Alfalfa plants comprising anintrogressed region comprising regions closely linked to or adjacent tothe genomic regions and markers provided herein and associated with ananthracnose disease resistance phenotype are also provided.

As an alternative to standard breeding methods of introducing traits ofinterest (e.g., introgression), biotechnological approaches can also beused. In one embodiment, marker-assisted technologies may be implementedto identify plants having a desired anthracnose disease resistancephenotype. Certain aspects of the invention also provide transgenicplant cells having stably integrated recombinant DNA constructs,transgenic plants and seeds comprising a plurality of such transgenicplant cells and transgenic pollen of such plants. In specificembodiments, recombinant DNA constructs integrated into an alfalfa plantaccording to the invention may possess enhanced resistance toanthracnose or other diseases. Alternatively, such plants may possessrecombinant DNA providing other traits “stacked” with an anthracnosedisease resistance trait of the invention. These stacked combinationscan be created by any method, including but not limited to, crossbreeding of transgenic plants or multiple genetic transformations.

An anthracnose resistant locus or allele at that locus may therefore beintroduced into any plant that contains any number or combination ofnon-transgenic or transgenic traits. Non-limiting examples of transgenictraits comprise herbicide tolerance, increased yield, insect control,fungal disease tolerance, virus tolerance, nematode tolerance, bacterialdisease tolerance, altered lignin composition or content, enhancedanimal and human nutrition, environmental stress resistance, increaseddigestibility, altered nitrogen utilization and improved seedproduction, among others. In one aspect, the herbicide tolerance isselected from the group consisting of glyphosate, dicamba, glufosinate,sulfonylurea, bromoxynil and norflurazon herbicides.

In particular further embodiments of the invention, methods are providedfor manufacturing seed that can be used to produce a crop of transgenicor non-transgenic plants with an enhanced trait, including resistancefor all of Race 1, Race 2, and Race 5 anthracnose as well as othertraits. In various methods of the invention, producing such plants maycomprise one or more of the following steps: (a) screening a populationof plants for an enhanced trait, where individual plants in thepopulation can exhibit the trait at a level less than, essentially thesame as, or greater than the level that the trait is exhibited incontrol plants, (b) selecting from the population one or more plantsthat exhibit the trait at a level greater than the level that said traitis exhibited in control plants, (c) collecting seed from a selectedplant. In particular embodiments, the method may comprise verifying thatone or more polymorphisms associated with the trait are present. In oneembodiment, identifying the presence of a trait may involve detectingthe presence of a marker polynucleotide molecule.

Other aspects of the various embodiments of the invention includeplants, seeds, and plant parts of a plant of the invention thatcomprises the anthracnose disease resistance trait described herein.Plant products and byproducts derived therefrom are also provided. Inspecific embodiments, such plant parts and derivatives may be defined ascomprising at least one marker polynucleotide molecule.

III. DEVELOPMENT OF DISEASE RESISTANT ALFALFA VARIETIES

For most breeding objectives, commercial breeders work within germplasmthat is “cultivated type” or “elite.” This germplasm is easier to breedbecause it generally performs well when evaluated for horticulturalperformance. Numerous elite alfalfa crop cultivated varieties(cultivars) have been developed. However, the performance advantage acultivated or elite germplasm provides can be offset by a lack ofallelic diversity. Breeders generally accept this tradeoff becauseprogress is faster when working with cultivated material than whenbreeding with genetically diverse sources.

The process of introgressing desirable resistance genes fromnon-cultivated lines into elite cultivated lines while avoiding problemswith linkage drag or low heritability is a long and often arduousprocess. Success in deploying alleles derived from wide sourcestherefore strongly depends on minimal or truncated introgressions thatlack detrimental effects and reliable assays that preferably replacephenotypic screens. Success is further defined by simplifying geneticsfor key attributes to allow focus on genetic gain for quantitativetraits such as disease resistance. Moreover, the process ofintrogressing genomic regions from non-cultivated lines can be greatlyfacilitated by the availability of informative markers.

One of skill in the art would therefore understand that the alleles,polymorphisms, and markers provided by the invention allow the trackingand introduction of any of the genomic regions identified herein intoany genetic background to which an alfalfa species can be crossed. Inaddition, the genomic regions associated with disease resistancedisclosed herein can be introgressed from one genotype to another andtracked phenotypically or genetically. Thus, Applicants' development ofmarkers for the selection of the disease resistance facilitates thedevelopment of alfalfa plants having beneficial phenotypes. For example,plants and seeds can be genotyped using the markers of the presentinvention in order to develop varieties comprising desired diseaseresistance. Moreover, marker-assisted selection (MAS) allowsidentification of plants which are homozygous or heterozygous thedesired introgression.

Meiotic recombination is essential for plant breeding because it enablesthe transfer of favorable alleles across genetic backgrounds, theremoval of deleterious genomic fragments, and pyramiding traits that aregenetically tightly linked. In the absence of accurate markers, limitedrecombination forces breeders to enlarge segregating populations forprogeny screens. Moreover, phenotypic evaluation is time-consuming,resource-intensive and not reproducible in every environment,particularly for traits like disease resistance. The markers provided bythe invention offer an effective alternative and therefore represent asignificant advance in the art.

IV. MOLECULAR ASSISTED BREEDING TECHNIQUES

Genetic markers that can be used in the practice of the presentinvention include, but are not limited to, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),simple sequence repeats (SSRs), simple sequence length polymorphisms(SSLPs), single nucleotide polymorphisms (SNPs), insertion/deletionpolymorphisms (Indels), variable number tandem repeats (VNTRs), andrandom amplified polymorphic DNA (RAPD), isozymes, and other markersknown to those skilled in the art. Vegetable breeders use molecularmarkers to interrogate a crop's genome and classify material based ongenetic, rather than phenotypic, differences. Advanced markertechnologies are based on genome sequences, the nucleotide order ofdistinct, polymorphic genotypes within a species. Such platforms enableselection for horticultural traits with markers linked to favorablealleles, in addition to the organization of germplasm using markersrandomly distributed throughout the genome. In the past, a prioriknowledge of the genome lacked for major vegetable crops that now havebeen sequenced. Scientists exploited sequence homology, rather thanknown polymorphisms, to develop marker platforms. Man-made DNA moleculesare used to prime replication of genome fragments when hybridizedpair-wise in the presence of a DNA polymerase enzyme. This synthesis,regulated by thermal cycling conditions that control hybridization andreplication of DNA strands in the polymerase chain reaction (PCR) toamplify DNA fragments of a length dependent on the distance between eachprimer pair. These fragments are then detected as markers and commonlyknown examples include AFLP and RAPD. A third technique, RFLP does notinclude a DNA amplification step. Amplified fragment length polymorphism(AFLP) technology reduces the complexity of the genome. First, throughdigestive enzymes cleaving DNA strands in a sequence-specific manner.Fragments are then selected for their size and finally replicated usingselective oligonucleotides, each homologous to a subset of genomefragments. As a result, AFLP technology consistently amplifies DNAfragments across genotypes, experiments and laboratories.

Polymorphisms comprising as little as a single nucleotide change can beassayed in a number of ways. For example, detection can be made byelectrophoretic techniques including a single strand conformationalpolymorphism, denaturing gradient gel electrophoresis, or cleavagefragment length polymorphisms, but the widespread availability of DNAsequencing often makes it easier to simply sequence amplified productsdirectly. Once the polymorphic sequence difference is known, rapidassays can be designed for progeny testing, typically involving someversion of PCR amplification of specific alleles, or PCR amplificationof multiple specific alleles.

Polymorphic markers serve as useful tools for assaying plants fordetermining the degree of identity of lines or varieties. These markersform the basis for determining associations with phenotypes and can beused to drive genetic gain. In certain embodiments of methods of theinvention, polymorphic nucleic acids can be used to detect in an alfalfaplant a genotype associated with disease resistance, identify an alfalfaplant with a genotype associated with disease resistance, and to selectan alfalfa plant with a genotype associated with disease resistance. Incertain embodiments of methods of the invention, polymorphic nucleicacids can be used to produce an alfalfa plant that comprises in itsgenome an introgressed locus associated with disease resistance. Incertain embodiments of the invention, polymorphic nucleic acids can beused to breed progeny an alfalfa plants comprising a locus associatedwith disease resistance.

Genetic markers may include “dominant” or “codominant” markers.“Codominant” markers reveal the presence of two or more alleles (two perdiploid individual). “Dominant” markers reveal the presence of only asingle allele. Markers are preferably inherited in codominant fashion sothat the presence of both alleles at a diploid locus, or multiplealleles in triploid or tetraploid loci, are readily detectable, and theyare free of environmental variation, i.e., their heritability is 1. Amarker genotype typically comprises two marker alleles at each locus ina diploid organism. The marker allelic composition of each locus can beeither homozygous or heterozygous. Homozygosity is a condition whereboth alleles at a locus are characterized by the same nucleotidesequence. Heterozygosity refers to different conditions of the allele ata locus.

Nucleic acid-based analyses for determining the presence or absence ofthe genetic polymorphism (i.e., for genotyping) can be used in breedingprograms for identification, selection, introgression, and the like. Awide variety of genetic markers for the analysis of geneticpolymorphisms are available and known to those of skill in the art. Theanalysis may be used to select for genes, portions of genes, QTL,alleles, or genomic regions that comprise or are linked to a geneticmarker that is linked to or associated with disease resistance.

As used herein, nucleic acid analysis methods include, but are notlimited to, PCR-based detection methods (for example, TaqMan assays),microarray methods, mass spectrometry-based methods and/or nucleic acidsequencing methods, including whole genome sequencing. In certainembodiments, the detection of polymorphic sites in a sample of DNA, RNA,or cDNA may be facilitated through the use of nucleic acid amplificationmethods. Such methods specifically increase the concentration ofpolynucleotides that span the polymorphic site, or include that site andsequences located either distal or proximal to it. Such amplifiedmolecules can be readily detected by gel electrophoresis, fluorescencedetection methods, or other means.

One method of achieving such amplification employs the polymerase chainreaction (PCR) using primer pairs that are capable of hybridizing to theproximal sequences that define a polymorphism in its double-strandedform. Methods for typing DNA based on mass spectrometry can also beused.

Polymorphisms in DNA sequences can be detected or typed by a variety ofeffective methods well known in the art including, but not limited to,those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015;5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876;5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039;7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of whichare incorporated herein by reference in their entirety. However, thecompositions and methods of the present invention can be used inconjunction with any polymorphism typing method to type polymorphisms ingenomic DNA samples. These genomic DNA samples used include but are notlimited to, genomic DNA isolated directly from a plant, cloned genomicDNA, or amplified genomic DNA.

For instance, polymorphisms in DNA sequences can be detected byhybridization to allele-specific oligonucleotide (ASO) probes asdisclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No.5,468,613 discloses allele specific oligonucleotide hybridizations wheresingle or multiple nucleotide variations in nucleic acid sequence can bedetected in nucleic acids by a process in which the sequence containingthe nucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Target nucleic acid sequence can also be detected by probe ligationmethods, for example as disclosed in U.S. Pat. No. 5,800,944 wheresequence of interest is amplified and hybridized to probes followed byligation to detect a labeled part of the probe.

Microarrays can also be used for polymorphism detection, whereinoligonucleotide probe sets are assembled in an overlapping fashion torepresent a single sequence such that a difference in the targetsequence at one point would result in partial probe hybridization(Borevitz, et al., Genome Res. 13:513-523, 2003; Cui, et al.,Bioinformatics 21:3852-3858, 2005). On any one microarray, it isexpected there will be a plurality of target sequences, which mayrepresent genes and/or noncoding regions wherein each target sequence isrepresented by a series of overlapping oligonucleotides, rather than bya single probe. This platform provides for high throughput screening ofa plurality of polymorphisms. Typing of target sequences bymicroarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122;6,913,879; and 6,996,476.

Other methods for detecting SNPs and Indels include single baseextension (SBE) methods. Examples of SBE methods include, but are notlimited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431;5,595,890; 5,762,876; and 5,945,283.

In another method for detecting polymorphisms, SNPs and Indels can bedetected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930;and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescentreporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ends of the probe. When the probe is intact, the proximity of thereporter dye to the quencher dye results in the suppression of thereporter dye fluorescence, e.g. by Forster-type energy transfer. DuringPCR forward and reverse primers hybridize to a specific sequence of thetarget DNA flanking a polymorphism while the hybridization probehybridizes to polymorphism-containing sequence within the amplified PCRproduct. In the subsequent PCR cycle DNA polymerase with 5′→3′exonuclease activity cleaves the probe and separates the reporter dyefrom the quencher dye resulting in increased fluorescence of thereporter.

In another embodiment, a locus or loci of interest can be directlysequenced using nucleic acid sequencing technologies. Methods fornucleic acid sequencing are known in the art and include technologiesprovided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience(Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-CORBiosciences (Lincoln, Neb.), NimbleGen Systems (Madison, Wis.), Illumina(San Diego, Calif.), and VisiGen Biotechnologies (Houston, Tex.). Suchnucleic acid sequencing technologies comprise formats such as parallelbead arrays, sequencing by ligation, capillary electrophoresis,electronic microchips, “biochips,” microarrays, parallel microchips, andsingle-molecule arrays.

V. ALFALFA VARIETY C0416C4164

The results of an objective evaluation of the C0416C4164 alfalfa varietyare presented below, in Table 1. Those of skill in the art willrecognize that these are typical values that may vary due to environmentand that other values that are substantially equivalent are within thescope of the invention.

TABLE 1 Phenotypic Description of C0416C4164 Alfalfa 1. FALL DORMANCYNAAIC Protocol-Regrowth Score Regrowth Score Check Varieties ModeratelyModerately Dormant Dormant Dormant (‘Excalibur’, (‘Saranac’ (‘Ranger’,Testing Date Application ‘Du Puits’, ‘WL 316’ ‘Arrow’, Institution Dateof Regrowth Variety ‘555’, ‘Legend’, ‘WL 317’, and Location Last CutScored C0416C4164 ‘Archer’) ‘G2852’) ‘WL325HQ’) LSD.05 CV x Forage Sep.14, 2016 Oct. 4, 2016 5.6 5.0 6.0 6.8 0.48 7.4 6.0 GeneticsInternational — West Salem,WI Forage Sep. 14, 2016 Oct. 4, 2016 5.4 5.06.0 7.0 0.49 7.5 5.9 Genetics — International Boone, WI 2. DISEASERESISTANC-Anthracnose (Race 1) (Colletotrichum trifolii) NAAIC ProtocolForage Genetics International-Nampa, ID-Greenhouse-2016 Resistance/ClassUnadjusted Number of Expected Syn. Gen. % Plants Variety Value TestedResistance Tested LSD .05 CV x C0416C4164 HR 59% Syn 1 48% 200 9.0%17.1% 37% 'Arc' HR 65% 54% 'Sarnac' S 2% 2% 3. DISEASERESISTANCE-Aphanomyces Root Rot (Race 1) (Aphanomyces euteiches) NAAICProtocol Forage Genetics International-West Salem, WI-Greenhouse-2016Resistance/Class Unadjusted % Number of Plants Variety Expected ValueSyn. Gen. Tested Resistance Tested C0416C4164 HR 61% Syn 1 57% 200'WAPH-1' R 50% 47% 'Saranac' S 1%  0% 4. DISEASE RESISTANCE-AphanomycesRoot Rot (Race 2) (Aphanomyces euteiches) NAAIC Protocol ForageGeneticsInternational-West Salem, WI-Greenhouse-2017 Resistance/ UnadjustedClass Syn. Gen. % Number of Variety Expected Value Tested ResistancePlants Tested LSD .05 CV x C0416C4164 HR 56% Syn 1 56% 200 9.6% 11.1%53% 'WAPH-5' R 50% 50% 'Saranac' S 1% 0% 'WAPH-1' S 1% 0% 5. DISEASERESISTANCE-Phytophthora Root Rot (Phytophthora megasperma f.medicaginis) NAAIC Protocol Forage Genetics International-West Salem,WI-Greenhouse-2016 Resistance/ Unadjusted Number of Class Syn. Gen. %Plants Variety Expected Value Tested Resistance Tested LSD .05 CV xC0416C4164 HR 57% Syn 1 53% 150 11.2% 13.3% 51% 'WAPH-1' R 55% 51%'Saranac' S 3% 4% 6. DISEASE RESISTANCE-Anthracnose (Race 5) NAAICProtocol Greenhouse-2016 Syn. Gen. Tested Unadjusted % Number of VarietyTested Resistance Plants LSD .05 CV x C0416C4164 Syn 1 71% 200 8.1%20.4% 25.9% 'Arc' 7% 'Saranac AR' 12% 'Saranac' 2%

VI. ALFALFA VARIETY H0415C4114

The results of an objective evaluation of the H0415C4114 alfalfa varietyare presented below, in Table 2. Those of skill in the art willrecognize that these are typical values that may vary due to environmentand that other values that are substantially equivalent are within thescope of the invention.

TABLE 2 Phenotypic Description of H0415C4114 Alfalfa 1. FALL DORMANCYNAAIC Protocol-Regrowth Score Regrowth Score Testing Check VarietiesInstitution Date Application Moderately Moderately and Date of RegrowthVariety Dormant Dormant Dormant Location Last Cut Scored H0415C4114(‘Archer’) (‘G2852’) (‘WL325HQ’) LSD .05 CV x Forage Sep. 8, 2015 Oct.6, 2015 5.5 5.3 6.0 6.9 0.49 7.0 4. Genetics 4 International — WestSalem, WI Forage Sep. 11, 2015 Oct. 2, 2015 5.8 4.9 6.0 6.9 0.32 4.6 4.Genetics 3 International — Boone, IA 2. DISEASE RESISTANCE-Anthracnose(Race 1) (Colletotrichum trifolii) NAAIC Protocol Forage GeneticsInternational-Nampa, ID Greenhouse-2015 Unadjusted Number ofResistance/Class Syn. Gen. % Plants Variety Expected Value TestedResistance Tested LSD .05 CV x H0415C4114 HR 71% Syn1 78% 200 9% 9% 76%'Arc' HR 65% 71% 'Sarnac' S 4% 4% 3. DISEASE RESISTANCE-Aphanomyces RootRot (Race 1) (Aphanomyces euteiches) NAAIC Protocol Forage GeneticsInternational-West Salem, WI Greenhouse-2015 Unadjusted Resistance/ClassSyn. Gen. % Number of Variety Expected Value Tested Resistance PlantsTested LSD .05 CV x H0415C4114 HR 66% Syn1 58% 200 5% 6% 62% 'WAPH-1' R50% 44% 'Saranac' S 1% 5% 4. DISEASE RESISTANCE-Aphanomyces Root Rot(Race 2) (Aphanomyces euteiches) NAAIC Protocol Forage GeneticsInternational-West Salem, WI Greenhouse-2015 Unadjusted Resistance/ClassSyn. Gen. % Number of Variety Expected Value Tested Resistance PlantsTested LSD .05 CV x H0415C4114 HR 58% Syn1 42% 200 2% 5% 41% 'WAPH-5' R50% 36% 'Saranac' S 1% 3% 'WAPH-1' S 0% 0% 5. DISEASERESISTANCE-Bacterial Wilt (Clavibacter michiganese) NAAIC ProtocolForage Genetics International-West Salem, WI-Field-2016 UnadjustedResistance/Class Syn. Gen. % Number of Variety Expected Value TestedResistance Plants Tested LSD .05 CV x H0415C4114 HR 77% Syn1 67% 200 13%20% 48% 'Vernal' R 42% 37% 'Sonora' S 1% 0% 6. DISEASERESISTANCE-Phytophthora Root Rot (Phytophthora megasperma f.medicaginis) NAAIC Protocol Forage Genetics International-West Salem, WIGreenhouse-2015 Unadjusted Resistance/Class Syn. Gen. % Number ofVariety Expected Value Tested Resistance Plants Tested LSD .05 CV xH0415C4114 HR 64% Syn1 59% 200 4% 6% 48% 'WAPH-1' R 55% 50% 'Saranac' S3% 3% 7. DISEASE RESISTANCE-Anthracnose (Race 5) (Colletotrichumtrifolii) NAAIC Protocol Forage Genetics International-West Salem, WIGreenhouse-2017 Unadjusted Resistance/Class Syn. Gen. % Number ofVariety Expected Value Tested Resistance Plants Tested LSD .05 CV xH0415C4114 R 44% Syn1 44% 200 17% 36% 30% 'Saranac AR' S 14% 14%'Saranac' S 0% 0% 'Arc' S 0% 0% 8. PEST AND DISEASE RESISTANCE-StemNematode (Ditylenchus dipsaci) NAAIC Protocol Forage GeneticsInternational-Nampa, ID-Greenhouse-2016 Unadjusted Resistance/Class Syn.Gen. % Number of Variety Expected Value Tested Resistance Plants TestedLSD .05 CV x H0415C4114 R 35% Syn1 29% 400 8% 13% 40% 'Vernema' R 60%49% 'Ranger' S 5% 7% 9. OTHER-RoundUp Ready Tolerance NAAIC ProtocolForage Genetics InternationalTouchet, WA-Greenhouse-2015 UnadjustedResistance/Class Syn. Gen. % Number of Variety Expected Value TestedResistance Plants Tested LSD .05 CV x H0415C4114 HT 89% Syn1 87% 800 4%3% 85% 'FGI-RR90' HT 90% 89% 'Saranac' S 0% 0%

VII. ADDITIONAL ALFALFA LINES

In one embodiment of the invention, a plant is provided that comprisesthe anthracnose resistance trait found in plants of which representativeseeds were deposited under American Type Culture Collection (ATCC)Accession No. PTA-124210. In another embodiment, a plant of theinvention is provided which is defined as sharing an ancestral geneticsource for the anthracnose resistance trait found in plants for whichsuch representative seed were deposited. In still yet another embodimentof the invention, a plant comprising the anthracnose resistance trait isthe alfalfa line C0416C4164 or a progeny thereof that inherited theanthracnose resistance trait therefrom.

Another aspect of the invention provides methods for crossing thealfalfa line C0416C4164 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of line C0416C4164, or can be used to produce hybrid alfalfaseeds and the plants grown therefrom. Hybrid seeds are produced bycrossing line C0416C4164 with second alfalfa parent line.

In another embodiment of the invention, a plant is provided thatcomprises the anthracnose resistance trait found in plants of whichrepresentative seeds were deposited under American Type CultureCollection (ATCC) Accession No. PTA-125043. In another embodiment, aplant of the invention is provided which is defined as sharing anancestral genetic source for the anthracnose resistance trait found inplants for which such representative seed were deposited. In still yetanother embodiment of the invention, a plant comprising the anthracnoseresistance trait is the alfalfa line H0415C4114 or a progeny thereofthat inherited the anthracnose resistance trait therefrom.

Another aspect of the invention provides methods for crossing thealfalfa line H0415C4114 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of line H0415C4114, or can be used to produce hybrid alfalfaseeds and the plants grown therefrom. Hybrid seeds are produced bycrossing line H0415C4114 with second alfalfa parent line.

In another aspect, the present invention provides a method of introgressing an anthracnose resistance trait into an alfalfa plant comprising:(a) crossing at least a first alfalfa line having increased anthracnoseresistance with a second alfalfa line to form a segregating population;(b) screening the population for anthracnose resistance; and (c)selecting at least one member of the population having an increased oraltered anthracnose resistance.

VIII. DEFINITIONS

The following definitions are provided to better define the presentinvention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cells of tissue culture from which alfalfa plants canbe regenerated, plant calli, plant clumps and plant cells that areintact in plants or parts of plants such as pollen, flowers, seeds,leaves, stems, and the like.

As used herein, the term “population” means a genetically heterogeneouscollection of plants that share a common parental derivation.

As used herein, the terms “variety” and “cultivar” mean a group ofsimilar plants that by their genetic pedigrees and performance can beidentified from other varieties within the same species.

As used herein, the term “transgenic plant” means a plant that compriseswithin its cells a heterologous polynucleotide. Generally, theheterologous polynucleotide is stably integrated within the genome suchthat the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette.

As used herein, the term “transgenic” means any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicorganisms or cells initially so altered, as well as those created bycrosses or asexual propagation from the initial transgenic organism orcell. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extrachromosomal) byconventional plant breeding methods (e.g., crosses) or by naturallyoccurring events such as random cross-fertilization, non-recombinantviral infection, non-recombinant bacterial transformation,non-recombinant transposition, or spontaneous mutation.

As used herein, the term “plant part” includes a cell, a seed, a root, astem, a leaf, a head, a flower, or pollen, as well as any other part orportion of a plant.

As used herein, an “allele” refers to one of two or more alternativeforms of a genomic sequence at a given locus on a chromosome.

A “Quantitative Trait Locus (QTL)” is a chromosomal location thatencodes for at least a first allele that affects the expressivity of aphenotype.

As used herein, a “marker” means a detectable characteristic that can beused to discriminate between organisms. Examples of such characteristicsinclude, but are not limited to, genetic markers, biochemical markers,metabolites, morphological characteristics, and agronomiccharacteristics.

As used herein, the term “phenotype” means the detectablecharacteristics of a cell or organism that can be influenced by geneexpression.

As used herein, the term “genotype” means the specific allelic makeup ofa plant.

As used herein, “elite line” or “cultivated line” means any line thathas resulted from breeding and selection for superior agronomicperformance. An “elite plant” refers to a plant belonging to an eliteline. Numerous elite lines are available and known to those of skill inthe art of alfalfa breeding. An “elite population” is an assortment ofelite individuals or lines that can be used to represent the state ofthe art in terms of agronomically superior genotypes of a given cropspecies, such as an alfalfa line. Similarly, an “elite germplasm” orelite strain of germplasm is an agronomically superior germplasm.

As used herein, the term “introgressed,” when used in reference to agenetic locus, refers to a genetic locus that has been introduced into anew genetic background, such as through backcrossing. Introgression of agenetic locus can be achieved through plant breeding methods and/or bymolecular genetic methods. Such molecular genetic methods include, butare not limited to, various plant transformation techniques and/ormethods that provide for homologous recombination, non-homologousrecombination, site-specific recombination, and/or genomic modificationsthat provide for locus substitution or locus conversion.

As used herein, the terms “recombinant” or “recombined” in the contextof a chromosomal segment refer to recombinant DNA sequences comprisingone or more genetic loci in a configuration in which they are not foundin nature, for example as a result of a recombination event betweenhomologous chromosomes during meiosis.

As used herein, the term “linked,” when used in the context of nucleicacid markers and/or genomic regions, means that the markers and/orgenomic regions are located on the same linkage group or chromosome suchthat they tend to segregate together at meiosis.

As used herein, “resistance locus” means a locus associated withresistance or tolerance to disease. For instance, a resistance locusaccording to the present invention may, in one embodiment, controlresistance or susceptibility to black rot.

As used herein, “resistance allele” means the nucleic acid sequenceassociated with resistance or tolerance to disease.

As used herein “resistance” or “improved resistance” in a plant todisease conditions is an indication that the plant is less affected bydisease conditions with respect to yield, survivability and/or otherrelevant agronomic measures, compared to a less resistant, more“susceptible” plant. Resistance is a relative term, indicating that a“resistant” plant survives and/or produces better yields in diseaseconditions compared to a different (less resistant) plant grown insimilar disease conditions. As used in the art, disease “tolerance” issometimes used interchangeably with disease “resistance.” One of skillwill appreciate that plant resistance to disease conditions varieswidely, and can represent a spectrum of more-resistant or less-resistantphenotypes. However, by simple observation, one of skill can generallydetermine the relative resistance or susceptibility of different plants,plant lines or plant families under disease conditions, and furthermore,will also recognize the phenotypic gradations of “resistant.”

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more,”unless specifically noted. The terms “comprise,” “have” and “include”are open-ended linking verbs. Any forms or tenses of one or more ofthese verbs, such as “comprises,” “comprising,” “has,” “having,”“includes” and “including,” are also open-ended. For example, any methodthat “comprises,” “has” or “includes” one or more steps is not limitedto possessing only those one or more steps and also covers otherunlisted steps. Similarly, any plant that “comprises,” “has” or“includes” one or more traits is not limited to possessing only thoseone or more traits and covers other unlisted traits.

IX. DEPOSIT INFORMATION

A deposit was made of at least 2500 seeds of C0416C4164 Alfalfa, asdescribed herein. The deposit was made with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209USA. The deposit is assigned ATCC Accession No. PTA-124210, and the dateof deposit was May 23, 2017. Access to the deposit will be availableduring the pendency of the application to persons entitled thereto uponrequest. The deposit will be maintained in the ATCC Depository, which isa public depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if nonviable during that period. Applicantdoes not waive any infringement of their rights granted under thispatent or any other form of variety protection, including the PlantVariety Protection Act (7 U.S.C. 2321 et seq.).

A deposit was made of at least 2500 seeds of H0415C4114 Alfalfa, asdescribed herein. The deposit was made with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209USA. The deposit is assigned ATCC Accession No. PTA-125043, and the dateof deposit was Apr. 3, 2018. Access to the deposit will be availableduring the pendency of the application to persons entitled thereto uponrequest. The deposit will be maintained in the ATCC Depository, which isa public depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if nonviable during that period. Applicantdoes not waive any infringement of their rights granted under thispatent or any other form of variety protection, including the PlantVariety Protection Act (7 U.S.C. 2321 et seq.).

X. EXAMPLES Example 1 Identification of Emerging Anthracnose Race

Low levels of anthracnose symptoms were observed in field trials ofalfalfa varieties resistant to Race 1 and Race 2 anthracnose. Thepathogen was cultured and used to inoculate Saranac AR seedlings(Saranac AR is an approved resistant line for both Race 1 and Race 2anthracnose). In this initial test, no resistant Saranac AR plants wereobserved, suggesting the development of a new race of the pathogen.

A screening of proprietary breeding populations for resistance to thenew pathogen was thus initiated. Although low levels of resistance werefound in a few breeding populations, almost all of these “resistant”plants succumbed to the disease 1-2 months later. This latent infectionand late symptom development on seedlings is very uncharacteristic forRace 1 and Race 2 anthracnose. These results indicated that previouslyknown resistance to anthracnose was not effective against the new race,identified as Race 5.

Example 2 Anthracnose Screening Assay

A systematic study was developed to assess the effect of severalvariables in the standard anthracnose screening process. Variablesevaluated included the use of inoculum from the new race of thepathogen, inoculum concentration, length of incubation time, and timingof selection of resistant plants. This led to the development of anassay that accurately identified anthracnose resistance, allowing forthe identification of plants comprising resistance to the newlyidentified anthracnose race.

To develop the assay, a needle inoculation procedure was performed. Inthe needle inoculation procedure, plants having stems of approximately10 inches or longer were selected. The stems were taped at least 8inches above the soil to prevent the disease from spreading into theroots. Inoculum was prepared by swirling 1 drop of tween with 100 mL ofdistilled water in a beaker. The swirled solution was then poured intodishes of anthracnose spores. A rubber scarper was used to incorporatethe spores into the solution. The spore containing solution was thenpoured back into the beaker through a filter. A concentration of conidiaof between 1×10⁶ and 2×10⁶ was confirmed. A full needle of solution wasthen inserted above the tape on the plants. A small amount of solutionwas injected into the stem. The plants were housed in a greenhouse andwatered on a routine watering schedule. Seven days after inoculation,the stems of the plants were assessed. A % susceptibility and resistancewas determined by examining and counting individual plants. Susceptibleplants demonstrated diamond lesion, shepherds, or spreading disease uponvisual inspection of a stem. Resistant plants demonstrated no spreadingdisease; in some instance they exhibited a scar from the site of needlepuncture.

An anthracnose race inoculation was also performed for anthracnose.Flats of approximately 14 day old seedlings (or seedlings ofapproximately 3 inches in height) were water and allowed today forapproximately 2 hours. A 1 L spray bottle containing an anthracnosesolution of 50,000 spores/L of distilled water was applied to the flatsof alfalfa until run-off, which was approximately 5-10mL per flat. Theflats were covered with a 1020 no hole flat and the flats were tapedtogether, and the tape was crimped for secure the edges of the flats.The flats were stored in a dark, room temperature area for 48 hours.After 48 hours, the flat covers were removed, and the plants were scoredaccording to CPR0057AZM Devitalization and Final Disposition. Plantsthat were free of disease were transferred to individual peat cups forbreeding purposes.

Example 3 Identification of Race 5 Anthracnose-Resistant Lines

In Year 1, phenotypic screen of fall dormancy 4-5 germplasm wasconducted, and a very low frequency (<1%) of plants resistant to Race 5anthracnose was observed in five breeding lines. Lines exhibiting lowlevels of resistance to Race 5 were advanced to the next cycle ofevaluation. Resistance to Race 5 anthracnose was confirmed, and escapeseliminated, with stem inoculations of these lines using Race 5anthracnose. A single cycle of phenotypic selection led to thedevelopment of novel breeding populations with >31% resistant plants.Segregation ratios during this breeding process suggest a single gene,with partial dominance that provides resistance to Race 1, Race 2 andthe new Race 5.

Example 4 Evaluation of Race 5 Anthracnose Resistance in IdentifiedLines

Seed was produced from several of the new Race 5-resistant breedingpopulations. Seed was harvested in late July which allowed planting inmultiple location forage yield trials in mid-August of Year 2. The Year3 growing season exhibited significant rainfall at the trial locationsand Race 5 anthracnose symptoms were very severe.

At one trial location, a fungicide study using broad spectrum fungicideHeadline™ (BASF, Research Triangle Park, N.C.) was conducted wherein thefungicide was applied to Race 5-susceptible varieties after each harvestduring the season, and compared to a non-sprayed control. In Year 3, a20-25% increase in forage yield with fungicide treatment wasdemonstrated in the third and fourth harvest, when anthracnose symptomsare typically most severe. In the fall of Year 2, established trials atmultiple locations demonstrated significant anthracnose symptoms earlyJuly to mid-October on all entries, except for the new Race 5-resistantexperimental varieties, which were disease free. At all three locationsthe new Race 5-resistant experimental varieties, conventional, RoundupReady, and HarvXtra exhibited significantly higher yield in the last twoharvests than their susceptible counterparts and all commercial controlvarieties.

The average yield advantage for Race 5-resistant varieties in these Year2 harvests was 15-20%. The disease resistance evaluations are summarizedin FIG. 1. In FIG. 1, 412M107 is a control variety with anthracnoseresistance typical of modern commercial cultivars.

Table 3 shows results from a Year 2 forage yield trial conducted atLocation 1. Check 1 is the average yield of 3 check varieties (AttentionII, Hi-gest360, and HyburFOrce 3400). Check 2 is the average yield of 2check vanities (54R02) and WL372HQ. In both Tables 3 and 4, “Conv”indicates experimental varieties without genetically engineered traitsand “RRA+H” idnicates experimental varieties with either the HarvXtraand/or Roundup Ready traits.

TABLE 3 Anthracnose Screen of Experimental Plants for Year 1 ForageYield Trials at Location 1. Anthracnose Screened Experimentals Comparedto GE Check Classes # Avg Yld Dis Scm T/A % Check Checks AnExpsExperimentals Cut 1 Cut 2 Cut 3 Cut 4 Cut 1 Cut 2 Cut 3 Cut 4 Check 1Conv 12 2.63 2.41 2.32 1.78 0.91 1.01 1.15 1.46 Check 2 RRA + H 8 2.562.29 2.15 1.63 0.94 0.95 1.00 1.28

Table 4 shows results from the Year 2 forage yield trial conducted atLocation 2.

TABLE 4 Anthracnose Screen of Experimental Plants for Year 2 ForageYield Trials at Location 2. Anthracnose Screened Experimentals Comparedto GE Check Gasses # Avg Yld Dis Scm T/A % Check Checks AnExpsExperimentals Cut 1 Cut 2 Cut 3 Cut 4 Cut 5 Cut 1 Cut 2 Cut 3 Cut 4 Cut5 Check 1 Conv 12 2.10 2.04 1.48 1.37 0.77 0.89 1.03 1.26 1.26 1 24Check 2 RRA + H 8 1.87 1.90 1.36 1.27 0.72 0.83 0.94 1.04 1.15 0.99

Example 5 Development of Race 5 Anthracnose-Resistant Lines

FIG. 2 shows a comparison of a founder population for race 5 anthracnose(RRL43A132, a susceptible plant) to derived populations having race 5anthracnose resistance introgressed in HarvXtra (H0415C4112, H0415C4114,H0415C4115, and H0415C4116).

Race 5 resistance is score from 0 to 4, wherein 0 indicated dead plants,and 4 indicates highly resistant plants. FIG. 2 shows the distributionof individual plants within each population. These populations have had1 cycle of recurrent selection for Race 5 anthracnose resistance. Plantsscored as a 3 or 4 were considered resistant.

FIG. 3A illustrates that all RRL43A132 plants were dead 13 days afterinoculation with Race 5 anthracnose. RRL43A132 is the source populationthat led to many of the derived anthracnose Race 5 HarvXtra resistantexperimentals.

FIG. 3B illustrates that H0416C4116 alfalfa 13 days after inoculationwith Race 5 anthracnose yielded a large percentage of survivors comparedto the founder population (this population has had 1 cycle of phenotypicselection for Race 5 anthracnose resistance).

Example 6 Anthracnose Race 5 Resistance Locus Identification

Molecular markers associated with a QTL conferring resistance toanthracnose race 5 were identified by associating genetic variationsobserved to resistant and susceptible individuals. Nine synthetic (Syn1)alfalfa populations were studied: three conventional populations(C0415C4152, C0415C4159, C0415C4360), three Roundup Ready populations(R0415C3354, R0415C4156, R0415C4355) and three HarvXtra populations(H0415C4112, H0415C4114, H0415C4115). Seeds were planted in a greenhouseand seedlings were evaluated for Anthracnose Race 5 resistance by themethod of Nichole O'Neill (NAAIC Standard Test, 1991). Each plant wasscored as phenotypically as ‘resistant’ or ‘susceptible’.

Genotyping-by-Sequencing (GBS) methods were used to generate SNP markersassociated with the anthracnose race 5 resistance QTL, based on thephenotypic evaluation performed above. DNA was extracted fromlyophilized leaf tissue using the DNeasy 96 Plant Kit (Qiagen, 69181)per manufacturer's instructions. Extracted DNA samples were quantifiedusing the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589) priorto normalization. GBS libraries were constructed by the Elshire et al.(2011) method with minor modification. 100 ng of DNA from each samplewas digested with ApeKI (New England Biolabs, R0643L) and ligated withT4 DNA ligase (NEB, M0202L) to a common adaptor and a unique barcodedadapter. Pooled samples were purified using the QIAquick PCRPurification Kit (QIAGEN, 28104) and quantified again with the Quant-iTPicoGreen dsDNA Assay Kit. Each pool was amplified with LibraryAmplification Readymix (KAPA Biosystems, KK2611) and two 5 uM primersusing the following PCR program: 5 min at 72° C., 30 s at 98° C., 10cycles (10 s at 98° C., 30 s at 65° C., 30 s at 72° C.), 5 min at 72° C.PCR products were purified using the QIAquick PCR Purification Kit. Eachlibrary was sequenced on the Illumina HiSeq4000.

Raw GBS data were processed using the SNP CROP pipeline v.3.0 (Melo etal., 2016). Alignment and localization of the anthracnose race 5resistance QTL was carried out using the publicly available Medicagotruncatula reference genome JCVI.Medtr.v4.20130313. Only bi-allelic SNPspresent in at least 50% of individuals in the mapping population wereconsidered in the mapping marker set, resulting in a total of 5065 SNPs.The statistical association analysis was carried out using a mixed modelframework (i.e. Q+K GWA mixed model) via the GWASpoly R package (Rosyaraet al., 2016). The results indicated the presence of a significantanthracnose race 5 resistance QTL located on chromosome 4. Table 5 showsSNP markers identified as being associated with anthracnose race 5resistance from a joint analysis of the HarvXtra populations.

TABLE 5 HarvXtra Joint Analysis Marker Results Marker ChromosomePosition (bp) −log(p-value) FG2196 4 14,130,374 5.55 FG2197 4 14,130,3865.55 FG2201 4 15,513,183 8.27 FG2208 4 16,945,124 6.37 FG2218 416,968,138 19.23 FG2226 4 17,411,203 42.00 FG2274 4 23,797,145 5.80FG2337 4 27,310,905 4.36

Table 6 shows SNP markers identified as being associated withanthracnose race 5 resistance from a joint analysis of the Roundup Readypopulations.

TABLE 6 Roundup Ready Joint Analysis Marker Results Marker ChromosomePosition (bp) −log(p-value) FG27232 4 19,197,732 13.13 FG2724 419,197,756 5.29 FG27251 4 19,197,832 6.57 FG27271 4 19,292,176 6.96

Table 7 shows SNP markers identified as being associated withanthracnose race 5 resistance from a joint analysis of the Conventionalpopulations.

TABLE 7 Conventional Joint Analysis Marker Results Marker ChromosomePosition (bp) −log(p-value) FG2218 4 16,968,138 5.98 FG27062 417,408,441 6.19 FG27232 4 19,197,732 9.98

Odds ratio (OR) is among the most commonly used statistical measures ofassociation or effect, particularly, when dependent variables arebinary, such as studies of association between risk factors and diseaseoutcomes. In this case, the OR is the odds of resistance in plantscomprising the SNP divided by the odds of resistance in plants notcomprising the SNP. The ORs were calculated for the SNP showing thehighest significance within each of the three sets of populations.

For the HarvXtra populations, FG2226 was observed to be the SNP with thehighest significance. The OR was calculated to be 102.5((122×126)/(10×15)), based on the data obtained in Table 8. Thus, theodds of resistance to anthracnose race 5 in plants with the allele atFG2226 is ≈102 times higher than the odds of resistance to anthracnoserace 5 in plants without the allele at FG2226.

TABLE 8 Confusion Matrix for HarvXtra Joint Analysis Based on FG2226Observed = Susceptible Observed = Resistant Predicted = Susceptible 12210 Predicted = Resistant 15 126

For the Roundup Ready populations, FG27251 was observed to be the SNPwith the highest significance. The OR was calculated to be 87.59((74×116)/(49×2)), based on the data obtained in Table 9. Thus, the oddsof resistance to anthracnose race 5 in plants with the allele at FG27251is ≈88 times higher than the odds of resistance to anthracnose race 5 inplants without the allele at FG27251.

TABLE 9 Confusion Matrix for Roundup Ready Joint Analysis Based onFG27251 Observed = Susceptible Observed = Resistant Predicted =Susceptible 74 49 Predicted = Resistant 2 116

For the conventional populations (C0415C4152, C0415C4159, C0415C4360),FG27251 was observed to be the SNP with the highest significance. The ORwas calculated to be 16.17 ((153×52)/(6×82)), based on the data obtainedin Table 10. Thus, the odds of resistance to anthracnose race 5 inplants with the allele at FG27251 is ≈16 times higher than the odds ofresistance to anthracnose race 5 in plants without the allele atFG27251.

TABLE 10 Confusion Matrix for Conventional Joint Analysis Based onFG27251 Observed = Susceptible Observed = Resistant Predicted =Susceptible 153 82 Predicted = Resistant 6 52

The locus identified encompasses 7.9 cM on chromosome 4 and correspondsto the interval 16,945,124 bp to 19,292,176 bp of the public physicalmap. Table 11 lists the significant markers found to be associated withresistance to anthracnose race 5.

TABLE 11 Highly Significant SNP Markers Associated with Anthracnose Race5 Resistance Marker SNP Public Position Genetic Map Sequence FavorableJCVI.Medtr.v4.20130313 Distance (cM) Marker (SEQ ID NO) Chr. Allele (bp)(From Li et al., 2013) FG2208 1 4 G 16,945,124 ≥24.405 FG2218 2 4 A16,968,138 FG27062 3 4 A 17,408,441 FG2226 4 4 A 17,411,203 FG27232 5 4G 19,197,732 FG27251 6 4 T 19,197,832 FG27271 7 4 A 19,292,176 ≤32.328

Example 7 Methods for Producing Anthracnose Race 5 Resistant Plants

Plants comprising broad-spectrum resistance to anthracnose may beproduced by crossing a plant comprising an allele conferring anthracnoserace 5 resistance, representative seed comprising said allele havingbeen deposited under ATCC Accession No. PTA-124210 or under ATCCAccession No. PTA-125043, with a second plant to produce a progeny plantcomprising said allele. Progeny plants may further be selected for thepresence of said allele conferring anthracnose race 5 resistance.Selecting for the presence of said allele may include detecting at leastone genetic marker within or genetically linked to an identified genomicregion flanked in the genome of said plant by a first identified markerlocus and a second identified marker locus. In certain examples, thisincludes detecting at least one polymorphism at a locus selected fromthe group consisting of FG2208 (SEQ ID NO: 1), FG2218 (SEQ ID NO: 2),FG27062 (SEQ ID NO: 3), FG2226 (SEQ ID NO: 4), FG27232 (SEQ ID NO: 5),FG27251 (SEQ ID NO: 6), and FG27271 (SEQ ID NO: 7).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

1. A synthetic population of Medicago sativa plants, wherein at least53% of the plants of said population comprises an introgressed alleleconferring to said plants increased resistance to Colletotrichumtrifolii Race 5 compared with a plant not comprising said allele,wherein a representative sample of seed comprising said allele has beendeposited under ATCC Accession No. PTA-124210 or under ATCC AccessionNo. PTA-125043.
 2. The synthetic population of Medicago sativa plants ofclaim 1, wherein said allele is partially dominant.
 3. The syntheticpopulation of Medicago sativa plants of claim 1, wherein said populationcomprises resistance to Colletotrichum trifolii Races 1, 2, and
 5. 4.(canceled)
 5. The synthetic population of Medicago sativa plants ofclaim 1, wherein said introgressed allele comprises the resistancehaplotype found in H0415C4112, H0415C4114, H0415C4115, or H0415C4116. 6.A population of seed that produces the synthetic population of Medicagosativa plants of claim
 1. 7.-9. (canceled)
 10. A method for producingMedicago sativa seed or plants with resistance to Colletotrichumtrifolii, comprising the steps of: a) crossing the synthetic populationof Medicago sativa plants of claim 1 with other plants from saidpopulation or with Medicago sativa plants of a different genotype; andb) selecting seed or plants from said crossing.
 11. The method of claim10, wherein selecting said seed or plants comprises identifying agenetic marker genetically linked to said allele.
 12. The method ofclaim 10, wherein selecting seed or plants comprises identifying agenetic marker within or genetically linked to a genomic region flankedin the genome of said seed or plants by first identified marker locusand second identified marker locus.
 13. The method of claim 10, whereinselecting seed or plants comprises detecting at least one polymorphismat a locus selected from the group consisting of first marker locus,second marker locus, and third marker locus. 14.-43. (canceled)
 44. Asynthetic population of Medicago sativa plants, wherein at least 53% ofthe plants of said population comprises a recombinant chromosomalsegment on chromosome 4, wherein said chromosomal segment comprises anintrogressed Colletotrichum trifolii race 5 resistance allele conferringto said plants increased resistance to Colletotrichum trifolii race 5compared to a plant not comprising said allele.
 45. The syntheticpopulation of Medicago sativa plants of claim 44, wherein saidColletotrichum trifolii race 5 resistance allele is located within achromosomal segment is flanked by marker locus FG2208 (SEQ ID NO: 1) andmarker locus FG27271 (SEQ ID NO: 7) on chromosome 4 in said plants. 46.The synthetic population of Medicago sativa plants of claim 44, whereinsaid recombinant chromosomal segment comprises a marker locus selectedfrom the group consisting of FG2208 (SEQ ID NO: 1), FG2218 (SEQ ID NO:2), FG27062 (SEQ ID NO: 3), FG2226 (SEQ ID NO: 4), FG27232 (SEQ ID NO:5), FG27251 (SEQ ID NO: 6), and FG27271 (SEQ ID NO: 7) on chromosome 4.47. The synthetic population of Medicago sativa plants of claim 44,wherein said Colletotrichum trifolii race 5 resistance allele is locatedwithin a chromosomal segment in the genome of said plant flanked by16,945,124 bp to 19,292,176 bp on the Medicago truncatula Mt4.0 map. 48.The synthetic population of Medicago sativa plants of claim 44, whereina sample of seed comprising said introgressed Colletotrichum trifoliirace 5 resistance allele was deposited under ATCC Accession No.PTA-124210 or under ATCC Accession No. PTA-125043.
 49. A method forproducing Medicago sativa plants with resistance to Colletotrichumtrifolii, comprising the steps of: a) crossing the synthetic populationof Medicago sativa plants of claim 44 with plants of said syntheticpopulation or with Medicago sativa plants of a different genotype toproduce progeny plants; and b) selecting progeny plants comprising saidallele.
 50. The method of claim 49, wherein selecting progeny plantscomprises detecting said allele flanked by marker locus FG2208 (SEQ IDNO: 1) and marker locus FG27271 (SEQ ID NO: 7). 51.-58. (canceled) 59.The synthetic population of Medicago sativa plants of claim 1, whereinthe Medicago sativa plants further comprise a transgene conferringherbicide tolerance to said plants.
 60. The synthetic population ofMedicago sativa plants of claim 59, wherein the herbicide tolerance isto a herbicide selected from the group consisting of: glyphosate,dicamba, glufosinate, sulfonylurea, bromoxynil and norflurazonherbicides.
 61. The synthetic population of Medicago sativa plants ofclaim 1, wherein the Medicago sativa plants further comprise a transgenethat confers an altered lignin composition phenotype to said plants.